Dependence of adenine production upon polyamine synthesis in cultured human lymphoblasts.
The exact source of de novo adenine produced by mammalian cells remain poorly understood, and this prompted the present study. Using a human lymphoblastoid cell line (WI-L2) deficient in adenine phosphoribosyltransferase (EC 2.4.2.7), we have quantitated the rate of adenine synthesis and the relative importance of the phosphorolysis of 5'-methylthioadenosine versus adenosine or 2'-deoxyadenosine in adenine generation. Dividing adenine phosphoribosyltransferase-deficient WI-L2 cells produced adenine at a rate of 0.27 nmol/mg protein/h. This represented approximately 10% of the rate of hypoxanthine production by WI-L2 cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) but was equivalent to the rate of 5'-methylthioadenosine synthesis by human lymphoblastoid CCRF-CEM deficient in 5'-methylthioadenosine, phosphorylase (5'-methylthioadenosine: orthophosphate methylthioribosyltransferase). Up to 97% of adenine, but not hypoxanthine, synthesis was inhibited dose-dependently by the S-adenosylmethionine decarboxylase-inhibitor methylglyoxal bis(guanylhydrazone) and also by spermidine and spermine, but was enhanced by putrescine. The addition of 2-fluoroadenine, a potent competitive inhibitor of methylthioadenosine phosphorylase (Ki = 0.43 microM) to adenine phosphoribosyl-transferase-deficient cells resulted in a progressive accumulation of 5'-methylthioadenosine in the culture medium, and up to an 85% decrease in adenine production at non-toxic concentrations. These results show that de novo adenine synthesis by dividing human cells is considerable, and that 85-97% derives from the cleavage of 5'-methylthioadenosine and hence from polyamine synthesis.
D000225:Adenine / Q000096:biosynthesis*; D000228:Adenine Phosphoribosyltransferase / Q000172:deficiency; D000241:Adenosine / Q000031:analogs & derivatives / Q000172:deficiency; D012437:Adenosylmethionine Decarboxylase / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D014644:Genetic Variation; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency; D007945:Leukemia, Lymphoid / Q000378:metabolism; D011073:Polyamines / Q000096:biosynthesis*; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency; D011700:Putrescine / Q000096:biosynthesis; D013095:Spermidine / Q000096:biosynthesis; D013096:Spermine / Q000096:biosynthesis; D013154:Spleen / Q000378:metabolism; D013872:Thionucleosides / Q000172:deficiency
D011073:Polyamines; D013872:Thionucleosides; D013096:Spermine; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D000228:Adenine Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D012437:Adenosylmethionine Decarboxylase; D000225:Adenine; D000241:Adenosine; D013095:Spermidine; D011700:Putrescine